Reliable determination of viral infection through analysis of biological specimens for infectious virus and viral antigens is important because subsequent medical treatment depends upon the ability to differentiate between infections caused by bacteria or viruses. The selection of appropriate medical treatment is predicated on correct diagnosis of etiologic agent(s) and the ability to determine the group to which said agent(s) belongs.
The rapid diagnosis of bacterial infection by analysis of biological specimens, preferably peripheral blood samples, through the process of lysis centrifugation is described in detail in U.S. Pat. Nos. 3,875,012, 3,928,139, 3,932,222, 4,131,512, and 4,212,948 which are incorporated herein by reference. The apparatus disclosed and methods applied provide a means to concentrate microbial pathogens from a large sample volume to a reduced sample volume by selective lysis of somatic cells which leaves microbial pathogen cells intact which sediment into a concentrated zone upon application of centrifugal forces. Microbial pathogens concentrated into the zone are detected through growth on enrichment media which promote cell replication and expression of specific characteristics which aid in microbial species identification.
Previous methods commonly employed for recovering infectious virus from samples of biological origin, specifically peripheral blood specimens, include contacting samples of anticoagulant-treated whole blood or whole blood processed to serum with indicator cells specifically susceptible to viral cytopathic effects. Viable virus particles contained in the blood or serum sample infect susceptible indicator cells over a typical period of from 12 to 38 days under favorable incubation conditions. Infected cells undergo virus-induced lysis which appears as a clear area on confluent host cell layers. The relative degree of viral infectivity in the sample is determined by quantitating the clear areas, which constitute the viral cytopathic effect (CPE). Direct placement of whole anticoagulated blood has, as a major disadvantage, presence of erythrocytes in vast excess over other cells, e.g., polymorphonuclear leukocytes (PMN), monocytes, and lymphocytes which are more likely to contain virus. As a result, erythrocytes interfere with intimate contact between PMN, monocyte or lymphocyte cells and indicator tissue culture cells and thereby block efficient passage of virus from somatic cells to indicator tissue culture cells. Use of coagulated blood, namely, the serum fraction, results in loss of somatic cells (e.g., PMN, monocytes, and lymphocytes) which contain virus, and therefore also results in loss of virus contained in the somatic cells.
A significant improvement in the application of whole blood or blood serum samples to determine viral infectivity consists of obtaining through centrifugation an enriched specimen of white blood cells composed particularly of leukocytes which are known to phagocytize or engulf virus particles. One process provides a "buffy coat" or mixed leukocyte cell layer from an anticoagulant-treated whole blood specimen. The mixed leukocyte cell population sediments slower than the erythrocyte cell population upon centrifugation and therefore can be found layered above erythrocytes. This layer of mixed leukocytes can be carefully removed by pipette aspiration and contacted with a susceptible cell population for detection of virus by cytopathic effect. A further improvement for processing biological specimens is the use of materials which create density gradients upon centrifugation such as Ficoll-Paque/Dextran as in the method of Howell, C. L. et al., Journal of Clinical Microbiology 1979, 10(4):533-537. The mixed leukocyte cell population is further separated in this process into lymphocyte, monocyte, and PMN sub-populations. It is well known that certain viruses such as, but not limited to, Cytomegalovirus, are preferentially associated with the PMN population, while others, such as, but not limited to, Adenovirus and Varicella, are more often associated with monocyte populations. The selective isolation of these populations during centrifugation offers an improvement over conventional centrifugation by enriching the quantity of virus-bearing cells recovered from fractionation of a peripheral blood specimen. Detection of virus by contacting aliquots of each enriched leukocyte fraction with susceptible cell systems and quantitating cytopathic effect has been improved by increasing the number variety, and frequency of recovered virus. However, the major disadvantage of both the "buffy coat" and density gradient centrifugation techniques is that several laborious, time-consuming steps are required with a relatively high level of technical skill to obtain suitable specimens. Unfortunately, the improved diagnostic capability realized with leukocyte population fractionation requires this level of effort. There is a need for a rapid method to detect virus or viral antigens in biological samples which does not require sophisticated, multi-step procedures to obtain suitable analytical specimens for virus detection systems, particularly tissue culture.